Plant Cell Physiol. 40(7): 668-674 (1999)
JSPP © 1999
Photosynthetic Adaptation to Salt Stress in Three-Color Leaves of a C4 Plant
Amaranthus tricolor
Yumei Wang 1 , Yu-Ling Meng 2 , Hiroshi Ishikavva3, Takashi Hibino 3 , Yoshito Tanaka3, Naosuke Nii 1
and Teruhiro Takabe 2 - 4
1
2
3
Faculty of Agriculture, Meijo University, Tenpaku-ku, Nagoya, Aichi, 468-8502 Japan
Research Institute of Meijo University, Tenpaku-ku, Nagoya, Aichi, 468-8502 Japan
Department of Chemistry, Faculty of Science & Technology, Meijo University, Tenpaku-ku, Nagoya, Aichi, 468-8502 Japan
We examined the photosynthetic adaptation mechanisms for salt stress in Amaranthus tricolor, which has
leaves with green, yellow and red regions, in relation to the
accumulation of glycinebetaine as osmoprotectants. The
content of Chi, especially of Chi b in the red and yellow
regions was 3~4% of that in the green region. The levels
of Chi proteins such as LHCII, PSI and PSII were significantly lower than those in the green region. However,
the contents of other photosynthetic proteins in these
regions seem to be relatively high. We observed the net
photosynthetic CO2 fixation activity in the red and yellow
regions which was about 40% of that in the green region.
Upon salt stress (0.3 M NaCl) for 5 d the levels of Chi, PSI,
PSII, ribulose 1,5-bis phosphate carboxygenase and oxygenase, and the CO2 fixation rate in the green region decreased by about 20~35% whereas those in the non-green
regions remained almost at the same levels. A. tricolor was
found to accumulates glycinebetaine, betainealdehyde dehydrogenase and choline monooxygenase at similar levels
in all three color regions and their contents increased upon
salt stress. These results suggest that the low capacity of
light harvesting in non-green regions would be favor of salt
stress since the photosynthetic components in these regions
were retained at relatively high levels under high salinity.
Key words: Amaranthus tricolor — Betainealdehyde dehydrogenase — C4 plant — Choline monooxygenase —
Glycinebetaine — Salt stress.
Low molecular weight organic solutes such as sugars,
some amino acids, and quaternary ammonium compounds
involved in cell metabolism are accumulated to counteract
abiotic stress (Csonka and Hanson 1991, Bohnert and
Jensen 1996, Ingram and Bartels 1996, Takabe et al. 1998,
Zhu et al. 1997). Glycinebetaine (GB) is a quaternary ammonium compound present in bacteria, cyanobacteria, algae, animals and several plant families, but is absent in
Abbreviations: A., Amaranthus; BADH, betaine aldehyde
dehydrogenase; CMO, choline monooxygenase; Cyt/, cytochrome/; GB; glycinebetaine; PC, plastocyanin; RuBisCO, ribulose 1,5-bis phosphate carboxygenase and oxygenase.
4
To whom correspondence should be addressed.
many important crop species (Rhodes and Hanson 1993).
In plants, GB is synthesized by a two-step oxidation of
choline, via betaine aldehyde, by a ferredoxin-dependent
choline monooxygenase (CMO) (Rathinasabapathi et al.
1997) and the NAD+-dependent betaine aldehyde dehydrogenase (BADH) (Weigel et al. 1986, Arakawa et al.
1987). In bacteria, the first step is catalyzed by a choline
dehydrogenase (Lamark et al. 1991). The BADH enzyme
is known in Chenopodiaceae, Gramineae, and Amaranthaceae (McCue and Hanson 1992, Ishitani et al. 1993,
Valenzuela-Soto and Munoz-Clares 1994). Upon salt and
water stresses, BADH accumulates concomitantly with the
accumulation of GB (McCue and Hanson 1992, Ishitani et
al. 1993). In Chenopodiaceae, BADH is predominantly
located in chloroplasts and light is required for the synthesis of GB (Rathinasabapathi et al. 1994). The CMO
enzyme catalyzing the first step of GB synthesis is not well
known, having so far been found only in Chenopodiaceae
(spinach and sugar beet) and Amaranthaceae (Rathinasabapathi et al. 1997, Russell et al. 1998). CMO cDNAs
were isolated from spinach and sugar beet which were
completely unrelated to the bacterial choline dehydrogenase and oxidase enzymes (Rathinasabapathi et al. 1997).
Amaranthus is a glycophyte and C4 dicotyledonous
mesophyte crop plant. Previous work has shown that GB
accumulates in Amaranthus hypochondriacus L. (A. hypochondriacus) (Valenzuela-Soto and Munoz-Clares 1994)
and A. caudatus (Russell et al. 1998). A. tricolor is an Asian variety of amaranthus which produces unique leaves
that consist of three distinct color regions: green apices,
yellow middle, and red basal regions. These three-color
leaves emerge during the later phase of development and
are produced continuously thereafter. The red and yellow
regions have been reported to have markedly low levels of
Chi and lack photosynthetic activity (MaCormac et al.
1997). Although the gene expression of amaranthus is
complex, A. tricolor is very similar to A. hypochondriacus
in most aspects of morphology, growth, and development
(Wang et al. 1992, McCormac et al. 1997). Therefore, it
was interesting to examine the photosynthetic adaptation
mechanisms for salt stress in A. tricolor, especially in the
non-green regions in relation to the accumulation of GB.
668
Photosynthetic adaptation to salt in Amaranthus
Materials and Methods
Plant material and growth conditions—Seeds of A. tricolor
were grown with vermiculite in pots filled with 1/2 strength MS
medium under controlled conditions (16 h white fluorescent light,
120//Em" 2 s"' at 25°C and 8h dark at 20°C with an average
humidity of 50%). The 1/2 strength MS medium was added in
appropiate times and changed every two-weeks. The fully expanded green or three-color leaves were used when they are approximately 70 to 100 mm in length which was about 3 months
after germination. To induce salt stress, plants were transferred to
a growth medium that contained 300 mM NaCl under the same
controlled conditions.
Measurement of proteins, pigment, and GB—One gram of
material from each leaf region was harvested. For the extraction
of pigments, the harvested material was ground in a mortar with
a pestle in 4 ml of 100% acetone, and centrifuged at 10,000 rpm
for 5 min in a Kubota microfuge (model 720) at 4°C. The precipitate was suspended in 4 ml of 80% acetone for green and yellow pigments and in 4 ml of water for red pigments. The extracts
were combined and the absorption spectra of pigments were
measured with a spectrophotometer (Shimadzu UV1600). Chi was
extracted with 80% acetone and the concentration was determined
according to the method of Mackinney (1941) as previously described (Hibino et al. 1991). The absorption spectra of leaf were
measured from the spectral reflectance and transmitance of leaf
using a portable spectroradiometer with integrating sphere (model
Li-1800, Li-Cor, Lincoln, U.S.A.). The percentage of absorbed
light (Y) at each wavelength was calculated as [1 —(reflectance+
transmitance)]. The absorbance of leaves was calculated as log[l/
0-y)].
For the protein extraction, the harvested material was ground
in a mortar with a pestle in 4 ml of 50 mM HEPES (pH 8.0),
0.02% y?-mercaptoeihanol, 1 mM EDTA, 10% glycerol and 0.5
mM phenylmethane-sulfonyl fluoride (buffer A). The homogenate
was centrifuged at 15,000 x g for 15 min, and the pellet and supernatant were used as the insoluble and soluble fractions of the
total proteins, respectively. Proteins were determined by the
method of Lowry et al. (1951). GB was extracted as described
previously and measured with an NMR (Nomura et al. 1995) or
TOF-MS (model KOMPACT MALDI IV tDE, Shimadzu/Kratos).
Measurements of gas exchange, CM fluorescence and redox
state of P700—Gas exchange was measured at 25 °C with an
open-loop photosynthesis system (model HCM-100, Walz, Effeltrich, Germany) under illumination with white light, 500fiEm' 2
s~'. Chi fluorescence was measured with a PAM fluorometer
(Walz, Effeltrich, Germany). The photooxidizable P700 was monitored in terms of the absorbance change at 830 nm in a PAM
fluorometer using the emitter-detector unit ED 800T (Schreiber et
al. 1988). Saturating light (500 fiE m~2 s"1) was applied to samples via a multibranched fiber optic system with a halogen lamp.
Linear electron transport was also measured with a PAM fluorometer as previously described (Tanaka et al. 1997). Pulse-modulated excitation was obtained from light emitter diode lamps with
a peak emission at 650 nm. Light intensity of actinic white light
was 15O^Em~ 2 s~'. Saturated white light pulse (800-ms pulse
length, 3,000//E m~2 s"1) was provided from a halogen lamp.
(Fm — F)/Fm' was used as the quantum yield of linear electrontransport activity under irradiation (Genty et al. 1989).
Other methods—SDS-PAGE and immunoblotting were carried out as previously described (Lee et al. 1997). Antisera raised
against the cucumber PsaD (Iwasaki et al. 1990), spinach plasto-
669
cyanin (PC) (Hibino et al. 1991), and Brasicca komatsuna cytochrome/(Cyt/) (Takabe et al. 1972) were prepared as described.
For construction of antibodies against spinach BADH and CMO,
spinach BADH and CMO genes were isolated and expressed in
E. coli of which detail will be described in a separate paper. The
band intensity was quantified by using an Image Master (Pharmacia Biotec, Sweeden).
Results
Pigment characterization of the three-color leaves—
Mature A. tricolor had green leaves at the basal portion,
but three-color leaves at the upper portion. As shown in
Fig. 1A, the three-color leaf had a green apex, yellow
middle, and red basal region. The fully expanded green leaf
had a green apex and middle, but red/green in basal
region. The size and location of the red/green region were
similar to the red color in the three-color leaf. The spectrum of Y (Fig. IB) which was calculated as [1—(reflectance + transmitance)], shows that the green and green/red
leaves absorb more than 80% of incident light over a
wavelength ranging from 350 nm to 700 nm, whereas the
absorption by red and yellow leaves was low at wavelength
larger than 600 and 500 nm, respectively. The absorption
spectrum (Fig. 1C) in the green region was similar to that in
the green/red region of the green leaf except the 550 nm
region which was absorbed by the vacuole-localized /?-cyanin (Piattelli et al. 1969, MaCormac et al. 1997). The absorbance around 600-650 nm was significantly lower in the
red and yellow regions than in the green region, suggesting
the low levels of Chi b in these regions. The absorption
spectrum for the green region of the fully expanded green
leaf (apical half of leaf) was essentially the same as that for
the green region of the three-color leaf (data not shown)
and hereafter only the results for the three-color leaf will be
shown.
The absorption spectra of the extracted pigments are
shown in Figs. 2A-D. The quantative analysis of Chi revealed that the Chi b content in the red and yellow regions
was extremely low, and the Chi a/b ratio in the red and
yellow regions was as high as, 5.4~5.9, whereas that in the
green region was about 2.5 (Table 1).
Effects of salt stress on the pigment composition in the
three color regions—After addition of 0.3 M NaCl to the
growth medium, the photosynthetic CO2 fixation rate and
Chi contents decreased gradually whereas those of the
control plants were almost the same (data not shown).
Table 1 shows that after a 5-day treatment of salt stress, the
Chls a and b in the green and green/red regions both decreased by about 35%. However, in the red and yellow
regions, Chi b remained at the same level and Chi a increased slightly. Consequently, Chi a/b ratios in the green
and green/red regions were constant, but those in the red
and yellow regions increased during salt stress. In Table 1,
the Chi a/b ratios higher than 6 are approximate values
670
Photosynthetic adaptation to salt in Amaranthus
Green/Red
400
500
600
700
Wavelength (nm)
800
Fig. 1 A; three-color and green leaves of A. tricolor. Both leaves were 10 cm in length. B; percent of absorbed light (Y) in each color
region which was calculated as [1 —(reflectance+transmitance)]. C; absorbance in each color region which was calculated as
because of the limited sensitivity of the absorption method
in high Chi a/b ratio (Mackinney 1941, Mullet et al. 1980).
Effects of salt stress on the photosynthetic activity in
three color regions—The red and yellow regions of Amaranthus have been reported to be photosynthetically inactive when measured by PAM Chi fluorescence (McCormac
et al. 1997). However, we observed the photosynthetic CO 2
fixation activity even in the red and yellow regions as
shown in Fig. 3A. Under non-stressed conditions, the CO 2
assimilation rate was the most active in the green regions
and the rates in the green/red, red, and yellow regions were
about 60, 43, and 45% of the green region, respectively
(Fig. 3A). Upon salt stress, the CO2 fixation rate in the
green and green/red regions decreased by 30 and 40%, respectively. On the other hand, the CO 2 fixation rate in the
red and yellow regions remained at similar levels even under salt stressed conditions.
The quantum yield of electron-transport activity was
similar in all three color regions as shown in Fig. 3B. Upon
salt stress, the quantum yields of electron-transport activity
did not change significantly. Relative contents of photooxidizable P700 in the green, green/red, red, and yellow
Table 1 Effects of salt on the levels of Chi in the green, green/red, red, and yellow regions of A. tricolor
Chi a (mg (gFW)-')
0 M NaCl 0.3 M NaCl
Chi b (mg (gFW)" 1 )
0 M NaCl 0.3 M NaCl
Chi 0 + 6 (mg (gFW)-')
0 M NaCl 0. 3 M NaCl
Chi a/b
0 M NaCl 0.3 M NaCl
Green
2.39
1.53
0.95
0.64
3.34
2.12
2.55
2.44
Green/red
2.45
1.52
0.94
0.60
3.39
2.12
2.61
2.53
Red
0.18
0.21
0.03
0.03
0.21
0.24
5.39
6.73
Yellow
0.23
0.26
0.04
0.04
0.27
0.30
5.92
7.81
Photosynthetic adaptation to salt in Amaranthus
1.5
Green / Red
Green
1.0
OMNad
I 0.5
Yellow
Red
0.5
0.4
0.3
).3 M NaCl
0.3 M NaCl
0.2
IMNaQ
0.1
0
400
500
600
700
800 400
500
600
700
800
Wavelength (nm)
Fig. 2 Absorption spectra of pigments extracted from equal
amounts in fresh weight (FW) of green, green/red, red, and
yellow regions of A. tricolor leaves. A. tricolor after treatment
with or without 0.3 M NaCl for 5 d as described in "Materials and
Methods".
0 0.3
0_O3
0 0.3
0JX3
0_0.3
G/R
""R"
NaCl (M)
regions of the non-stressed plants were 100, 70, 30, and 25,
respectively (Fig. 3C). Upon salt stress, the contents of
photooxidizable P700 in the green and green/red regions
decreased by 25 and 10%, respectively whereas those in the
red and yellow regions remained at the similar levels.
Essentially similar-results were obtained for the PSII contents (Fig. 3D). Under non-stressed conditions, the (Fm—
Fo) values in red and yellow regions were about 37% of
that in the green region, suggesting the reduced levels of
active PSII in non-green regions. The decrease of (Fm —
Fo) upon high salinity in the green and green/red regions
were due to both the decrease of F m and the increase of
Fo> indicating the damage of acceptor and donor sides of
PSII.
Effects of salt stress on the level of photosynthetic
proteins in three color regions—We examined the change
of protein level upon the salt stress. Proteins were analyzed by SDS-PAGE followed by Coomassie blue staining
(Fig.4A, B) or by immunoblotting (Fig.4C-E). As shown
in rightward arrowhead in Fig. 4A, the level of LHCII
polypeptides was significantly lower in the red and yellow
regions than in the green region which is consistent with the
results of Chi contents (Table 1). The level of PsaD subunit
of PSI in the red and yellow regions was about 30% of that
in the green region (Fig. 4C), which is a reduction similar to
that observed in the P700 content (Fig. 3C). The levels of
0 0.3
0 0.3
G
0 0.3
0 0.3
G/R
R
NaCl (M)
0 0.3
Y
0 0.3
0 0.3
0 0.3
0 0.3
NaCl (M)
0 0.3
671
0 0.3
NaCl (M)
Fig. 3 Changes of photosynthetic parameters in each color region of the leaves treated with or without 0.3 M NaCl for 5 d. A;
photosynthetic CO2 fixation rate. B; quantum yield (4F/F m ') of linear electron transport. C; photochemically oxidizable P700. D; the
difference of fluorescence intensity between maximum (Fm) and minimum (Fo), a parameter reflecting photochemically active PSII. G,
green region; G/R, green/red region; R, red region; Y, yellow region.
Photosynthetic adaptation to salt in Amaranthus
672
(C) Psa D
1 2
3 4
5
6 7
(A) Insoluble fractions
12 3 4 5 6 7 8
30
20
14.4
0 0.3 0 0.3 0 0.3 0 0.3
G
G/R
R
Y
NaCl(M)
(D)Cyt./
1
2 3 4 5 6 7 8
Z
Q I).3 0 0 3 0 0 3 0 » 3
G
G/R
R
Y
NaCI (M)
(B) Total fractions
12 3 4 5 6 7 8
0 0.3 0 0.3
G
G/R
0 0.3
R
(E)PC
12 3 4 5
0 0.3
Y
Fig. 5 Changes of GB contents during the salt stress. A. tricolor
was treated with or without 0.3 M NaCI for 5 d. Equal amounts of
three-colored leaves in fresh weight were harvested and their GB
contents were mesured as described in "Materials and Methods".
G, green region; G/R, green/red region; R, red region; Y, yellow
region.
6 7 8
indicate that the level of Chl-binding proteins in the red
and yellow regions was significantly reduced, whereas those
of other proteins such as C y t / a n d RuBisCO were similar
in all three color regions. Under salt-stressed conditions,
0.3 0 0 .O
G/R
K
NaCI (M)
0 0.3 0 0 3 0 0.3 0 0.3
G
G/R
R
Y
NaCI (M)
Fig. 4 Effects of salt stress on the levels of photosynthetic
proteins in each color region of the leaves. A. tricolor was treated
with or without 0.3 M NaCI for 5 d. In A, C, and D, the insoluble
fractions were isolated from each color region and equal amounts
of proteins (100 /Jg) were analyzed in each lane by SDS-PAGE
followed by Coomassie blue staining (A) or by immunoblotting
against PsaD (C) and C y t / (D). In B and E, the total protein
fractions were analyzed by SDS-PAGE followed by Coomassie
blue staining (B) or by immunoblotting against spinach PC (E). In
A, LHCII and RuBisCO bands are shown by rightward and
leftward arrowheads, respectively. In B, RuBisCO bands are
shown in arrowhead. G, green region; G/R, green/red region; R,
red region; Y, yellow region.
LHCII and PsaD did not change significantly upon salt
stress. On the other hand, the levels of both C y t / a n d PC
in the red and yellow regions were relatively high although
they were lower than that in the green region (Fig. 4D, E).
These levels did not change upon salt stress. The CO2
fixation enzyme, ribulose 1,5-bisphosphate carboxygenase
and oxygenase (RuBisCO), also was present in a similar
level in all three color regions (Fig.4B). The level of
RuBisCO in the green and green/red regions decreased
20% upon salt stress whereas that in the red and yellow
regions remained at almost the same level. These results
A) BADH
1 2 3 4 5 6 7 8
9 10
0 0.3 0 0.3 0 0.3 0 0.3 0 0.3
G
G/R
R
Y
Root
NaCI (M)
B) CMO
1 2 3 4 5 6 7 8
9 10
0 0.3 0 0.3 0 0.3
R
Y
Root
NaCI (M)
0 0.3 0 0.3
G
G/R
Fig. 6 Changes of BADH and CMO during salt stress. A. tricolor was treated with or without 0.3 M NaCI for 5 d. Equal
amounts in fresh weight of three-colored leaves were harvested
and analyzed by SDS-PAGE followed by immunoblotting against
BADH (A) and CMO (B). G, green region; G/R, green/red
region; R, red region; Y, yellow region.
Photosynthetic adaptation to salt in Amaranthus
significant amounts of RuBisCO were obtained in the insoluble membrane fractions in the red and yellow regions
(leftward arrowhead in Fig.4A).
Effects of salt stress on the expression of GB synthesis
genes—Previously, GB has been shown to accumulate in
some Amaranthaceae (Valenzuela-Soto and Munoz-Clares
1994, Russell et al. 1998). However, it was unknown whether the three color A. tricolor accumulates GB, especially
in the non-green regions. In this study, we found that
A. tricolor accumulates GB in the three-color regions. GB
contents in the green, green/red, red, and yellow regions
were similar (Fig. 5). Upon salt stress, the levels of GB increased about 3~4 fold in all three-color regions. The
Western blotting experiments indicated that the BADH
content is similar in all three color regions (Fig. 6A), and
the level increased about 1.3-fold upon salt stress. The
CMO enzyme was also detected in all three-color regions of
A. tricolor (Fig. 6B), and the level increased 3~4 fold upon
salt stress. The GB level in the salt-stressed root was significantly low (Fig. 5). The level of BADH in the root was
similar to that in the leaf (Fig. 6A), but the level of CMO
was significantly reduced in the root (Fig. 6B).
Discussion
Data presented here clearly show that A. tricolor have
a significant level of CO2 fixation activity even in the red
and yellow regions (Table 1, Fig. 3A). Since the reduced Fd
is required for CMO (Rathinasabapathi et al. 1997), it is
reasonable that the photochemically active red and yellow
leaves could acumulate GB. These results are different
from the previous report (McCormac et al. 1997), although
the Chi content was similar. This discrepancy may be due
to the different light conditions for growth and/or measurement with the PAM fluorometer.
The high Chi a/b ratio (Fig. 4) and low levels of Chi in
the red and yellow regions suggest that the supply of Chi,
especially Chi b was significantly reduced in the red and
yellow regions. The reduced levels of Chi b would cause
almost the absence of LHCII and the significant decrease
of PSI and PSH contents (Fig. 3, 4). However, other
components for photosynthesis such as Cyt/, PC, and
RuBisCO were at relatively high levels in the red and yellow
regions (Fig. 4). These results suggest that the light harvesting capacity in the red and yellow regions were significantly low. The different light harvesting capacity between the green region and the red and yellow regions
would cause the different response for salt stress. Under
salt-stress conditions, the electron transport system would
become over-reduced because of inhibition of electron
consumption due to stomata closure. Then, active oxygen
will be produced, which causes the inactivation of enzymes
and degradation of proteins as observed in the green and
green/red leaves. However, if light harvesting was low, the
673
production of active oxygen will be reduced and the inhibition of enzymes and degradation of proteins will be
reduced as observed in the red and yellow leaves. This
suggests that the red and yellow regions could be more salt
tolerant, although they had low CO2 fixation activity.
Under salt-stressed conditions, considerable amounts of
RuBisCO were observed in the thylakoid membrane fractions only in the red and yellow regions (Fig. 4A). Since the
CO2 fixation rate in the salt-stressed red and yellow regions
was retained at a level similar to that in the non-stressed
regions, the thyalkoid bound RuBisCO might play important physiological roles, such as direct utilization of
reducing power or light dependent activation via conformational change. These points remain to be clarified.
GB is believed to serve as a nontoxic solute for
cytoplasmic osmoregulation and a protectant against the
damaging effects of salt on proteins and membranes (Csonka
and Hanson 1991, Rhodes and Hanson 1993, Takabe et al.
1998). The transformants that produce small amounts of
GB have shown to confer salt tolerance (Hayashi et al.
1997, Takabe et al. 1998). Since the GB level increased
under salt-stressed conditions in all three color regions of
A. tricolor (Fig. 5), the increased GB might function as an
osmoprotectant. The accumulation levels of GB were similar in all three color regions of A. tricolor leaves (Fig. 5)
which suggests that upon salt stress, GB synthesis occurs in
all three color regions using photochemicaly reduced Fd
even though photosynthetic activity was quite different.
This indicates that since fewer electrons were produced for
Fd reduction in the red and yellow regions, larger fractions
of electrons in reduced Fd were used for the synthesis of
GB in the red and yellow regions compared with the green
region. The utilization of electrons in the reduced Fd for
GB production could also contribute to the decrease of
over-reduction of photosystem and consequently the
decrease of active oxygen production in the red and yellow
regions.
The GB content in the root was markedly lower than
that in the leaves. Although the level of BADH in the root
was similar to that in the leaf, the level of CMO was quite
low. Therefore, the low level of GB in the root was due to
the low level of CMO. This is in sharp contrast to the
findings in sugar beet in which CMO was expressed in the
root and GB accumulated in the root (Russell et al. 1998).
Osmoprotectant(s) other than GB might accumulate in the
root upon salt stress and requires further study. The regulation of gene expression of CMO and physiological roles
of BADH in the root are interesting subjectes to be
clarified.
674
Photosynthetic adaptation to salt in Amaranthus
This work was supported in part by Grants-in-Aid for
Scientific Research from the Ministry of Education, Science and
Culture of Japan, from the High-Tech Research Center of Meijo
University, and from the Scientific Frontier Project of Meijo
University.
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(Received February 9, 1999; Accepted April 16, 1999)